The aims of the proposed research are to characterize and elucidate the mechanisms by which hormones whose actions are mediated by the adenylate cyclase-cAMP-protein kinase system affect mechanical function of cardiac myocytes. This will involve measurements of changes in the Ca2+ sensitivity of isometric tension, shortening velocity, and rate of relaxation of enzymatically isolated, receptor-coupled, permeabilized cardiac myocytes in response to stimulation with hormone receptor agonists, antagonists, and putative second messengers. Hypotheses to be tested are: (i) beta-adrenergic dependent phosphorylation of troponin I is responsible for an observed decrease in the Ca2+ sensitivity of tension generation and may also account for an increase in the rate of tension relaxation, (ii) the effects of muscarinic and adenosine receptor agonists to decrease heart rate and antagonize the positive inotropic effect of beta-adrenergic stimulation may be explained, in part, by inhibition of the adenylate cyclase-cAMP-kinase system and dephosphorylation of myofibrillar proteins, and (iii) histamine and opioid receptor agonists activate the adenylate cyclase second messenger system leading to alterations in mechanical function which are similar but not identical to those observed upon beta-adrenergic receptor stimulation. To test these hypotheses alterations in mechanical data will be related to changes in autoradiographs of SDS gels from myocytes obtained following hormone receptor stimulation or upon exposure to second messengers. Comparison of alterations in mechanical properties and protein phosphorylations upon second messenger versus hormone receptor stimulation will help identify the second messenger system responsible for the hormone effects. Once an alteration in function has been correlated with phosphorylation of a myofibrillar protein, the phosphorylated protein will be selectively extracted from single cardiac myocytes and replaced with a non-phosphorylated form of that protein to establish whether altered function upon hormonal stimulation can be explained on the this basis. These experiments should provide new information regarding the molecular mechanisms by which hormone receptor stimulation leads to alterations in myocardial function and will be of use in defining potential clinical therapies for diseased myocardium.